The sirtuins are conserved NAD+-dependent protein deactylases that contribute to the regulation of cellular pathways and functions associated with aging and age-related diseases. The Saccharomyces cerevisiae (budding yeast) Sir2 protein was the first sirtuin to be described and was originally characterized as a factor required for transcriptional silencing at the silent mating-type loci and telomeres, and then later characterized as histone deacetylase. Histone deacetylation at the rDNA locus and sub-telomeric repeats has been linked to the regulation of replicative lifespan (RLS) of yeast mother cells, which is defined as the number of divisions a mother performs before senescing. Deletion of SIR2 shortens RLS and overexpression extends lifespan, presumably through its function at the rDNA and telomeres. ChIP-sequencing analysis of the yeast sirtuins revealed that Sir2 and its paralog, Hst1, are surprisingly co-enriched on the open reading frames of multiple genes involved in glycolysis, fermentation, translation, and cell wall biosynthesis, all processes that ae highly expressed in exponentially growing glucose cultures, but are then repressed when cells enter the diauxic shift, the period in growth when yeast cells shift their metabolism from glycolysis/fermentation toward the TCA cycle/respiration. In preliminary data, Sir2 and Hst1 are both required for efficiently, and specifically repressing glycolytic genes with Sir2 and Hst1 bound to the ORF. This is very intriguing because in mammals, the Sir2 homologs SIRT1 and SIRT6 have both been implicated in controlling glucose metabolism in order to maintain homeostasis. I propose that the regulation of glycolytic genes in yeast cells contributes to the control of RLS. The first specific aim is focused on determining how Sir2 and Hst1 repress transcription of glycolytic genes when they are bound to the ORF. The second specific aim is designed to test whether Sir2 and Hst1 control metabolic homeostasis in a manner similar to mammalian sirtuins, and the third specific aim is focused on the consequences of rapid Sir2 protein turnover during replicative aging on glycolytic gene expression, and figuring out how the age- associated decline of Sir2 protein impacts the distribution of Sir2 across the various competing chromatin loci. The goal is to establish glycolytic gene regulation (repression) as a critical aging-related function of Sir2 and its close paralog, Hst1. Confirmation of this type of regulation would provide a direct evolutionary link between yeast and humans, making it possible to study sirtuin metabolism regulation in yeast, and translate the findings to mammals.